Identification of hemocyte types and characterization of their immune function in the house fly based on morphological observation and single-cell RNA sequencing

Hemocytes represent a pivotal element of insect defense against pathogens through the mechanisms of cellular and humoral immunity. However, hemocyte types and functions exhibit variation across different insect taxa, leading to the lack of a standardized classification system for insect hemocytes. To gain insight into the immune mechanisms of the house fly Musca domestica, we used a combination of morphological observations and single-cell RNA sequencing techniques (scRNA-seq) to taxonomically characterize house fly larval hemocytes and analyze their immune function. As a result, five different types of hemocytes were identified from house fly larvae, granulocytes (GR), spherulocytes (SP), plasmatocytes (PL), prohemocytes (PR), and oenocytoids (OE). On the basis of microscopic observations, flow cytometry analysis and gene expression profiles, the immune functions of house fly hemocytes were hypothesized to be as follows: the GRs are responsible for phagocytosis, the SPs are highly expressive of lectins, and the PLs are highly expressive of antimicrobial peptides (AMPs) and are involved in nodule formation, the PRs act as progenitor cells and retain the differentiation potential of stem cells, and the OEs are involved in melanization reactions mainly through the expression of phenoloxidase (PO). Based on the scRNA-seq data, the marker genes for each type of hemocyte were identified. The present study unveils the heterogeneity of house fly hemocytes in terms of morphology, gene expression characteristics, and function, thereby establishing the foundation for an in-depth understanding of the immune mechanisms in house fly.